Field-realistic concentrations of a neonicotinoid insecticide influence socially regulated brood development in a bumblebee

The systemic neonicotinoid insecticides are considered as one of the key culprits contributing to ongoing declines in pollinator health and abundance. Bumblebees are among the most important pollinators of temperate zone plants, making their susceptibility to neonicotinoid exposure of great concern. We report that bumblebee (Bombus terrestris) colonies exposed to field-realistic concentrations of the commonly used neonicotinoid Imidacloprid grew slower, consumed less food, and produced fewer workers, males and gynes, but unexpectedly produced larger workers compared to control colonies. Behavioural observations show that queens in pesticide-treated colonies spend more time inactive and less time caring for the brood. We suggest that the observed effects on brood body size are driven by a decreased queen ability to manipulate the larva developmental programme. These findings reveal an intricate and previously unknown effect of insecticides on the social interactions controlling brood development in social insect colonies. Insecticide influences on the social mechanisms regulating larval development are potentially detrimental for bumblebees, in which body size strongly influences both caste differentiation and the division of labour among workers, two organization principles of insect societies.

Introduction
Anthropogenic stressors are a key driver of species decline, in part because they negatively affect underlying behavioural and physiological processes that are fundamental to fitness. Pesticides are considered among the most destructive stressors for insects. Their effects, including at sublethal, field-realistic levels, can ultimately reduce fitness through a suite of more subtle effects, many of which cannot be detected by standard practices for assessing pesticide toxicity.
Neonicotinoids are neurotoxins that bind to nicotinic acetylcholine receptors (nAChR), leading to paralysis and death of the exposed insect [25][26][27]. Their systemic form of action allows them to be rapidly absorbed by vegetal tissues and transported to most or all parts of the treated plant, including pollen and nectar in floral tissues [28,29]. Due to their high efficacy, low toxicity to vertebrates and allegedly low environmental contamination, neonicotinoids have become the most widely sold class of insecticides in the world [26,30]. Although their high toxicity enables applying neonics at lower amounts relative to other classes of insecticides, there is a growing body of evidence for their lethal and sublethal effects on nontarget insects, including pollinators [31]. These include evidence for negative influences on fertility [32], foraging activity and learning performance [33,34]. This evidence for non-specific toxicity has led to regulatory bans on the use of three commonly used neonic formulations, clothianidin, imidacloprid (IMD) and thiamethoxam, in the European Union and Canada [24,25]. The harmful effects of neonics may vary among species, including among species of bumblebees (Bombus spp. [22,35,36]).
Bumblebees are among the most important pollinators in temperate zones, and several species are reared at industrial scales for the purpose of commercial crop pollination, largely in greenhouses [37]. Studies on the effects of neonics on bumblebee biology have revealed a broad spectrum of sublethal negative effects that include reduced food consumption and nest growth [32,38], colony initiation [39,40] and interactions among nestmates [41], and compromised foraging and homing abilities [42][43][44]. Their canonical effects on the cholinergic system cannot easily explain some of these harmful effects. For example, there is evidence that exposure to neonics resulted in attenuated immune defense [45] and reduced fertility [38,[46][47][48].
Most bumblebee species have an annual life cycle wherein a new colony is founded by a single-mated queen that recently emerged from overwintering [49,50]. Once the first cohort of offspring is produced (which typically develop into worker bees), queens progressively feed them at the larval stage. Later on, when these first worker bees emerge as adults, the colony enters its eusocial stage, which is characterized by overlapping generations of a mother queen and her daughter worker bees, collective care for the brood, and a clear reproductive division of labour [50]. In the presence of workers, queens reduce the time they allocate to activities such as brood feeding, foraging and nest maintenance, and instead focus mostly on reproduction-related activities [51][52][53]. Workers, in turn, take over most tasks for the nest [54]. The division of labour among workers relates more to body size than to age, with larger worker bees showing morphological and physiological characteristics that appear to make them better fitted for foraging activities ( [55][56][57]; for a recent review see [58]). There is some evidence suggesting that smaller individuals are better in some functions, such as starvation resistance [59,60]. Task performance is flexible in bumblebees, with many workers switching between foraging and nursing activities, even within the same day [57,61]. The queen manipulates offspring development such that queen-reared workers develop over less time, are typically smaller bodied, and are not likely to develop into gynes [52,58]. Accordingly, worker body size is typically smaller at earlier stages of colony development, when queens primarily care for brood, then grow larger along with the emergence of workers that take on brood care activities [53,62].
Here, we explored how exposure to the widely used insecticide Imidacloprid (IMD) influences the dramatic changes in social organization that occur at the early stages of nest initiation in bumblebees. Specifically, we tested the hypothesis that chronic dietary exposure to field-realistic doses of IMD influences brood care behaviour, and thus brood development, in ways that ultimately influence the growth of incipient colonies.

Methods (a) Bumblebee colonies
Fourteen incipient B. terrestris colonies were purchased from Polyam Pollination Services (Yad-Mordechai, Israel). Each colony included a queen, 5-10 workers and brood at various stages of development. Upon arrival to our Bee Research Facility (at the E. Safra Campus of the Hebrew University of Jerusalem; Jerusalem, Israel), the entire colonies and wax combs were transferred to fresh wooden nesting boxes (21 cm × 21 cm × 12 cm) equipped with a top, side and front walls made of transparent Plexiglas enabling clear view of the inner parts of the nest. At this stage, the colonies were standardized to only include a queen, four workers and approximately 14 cm 2 of brood area that included offspring at all stages of development. The four workers were tagged with numbered plastic discs glued to their thoraces (Opalith tags; Christian Graze KG, Germany), allowing for individual identification. Additional information is provided in the electronic supplementary information.

(b) Experimental set-up
Incipient colonies were randomly assigned to one of three treatments: 'Control' treatment, which included five colonies provisioned with sugar syrup with no IMD; the 'IMD1' treatment included five colonies fed with sugar syrup in which we dissolved 1 µg l −1 of IMD; and the 'IMD10' treatment group included four colonies fed with sugar syrup in which we dissolved 10 µg l −1 of IMD. Previous studies have shown that these IMD doses are field-realistic (for example: [63][64][65][66][67]). The IMD stock solution (10 mg l −1 ) was produced by diluting 1 mg of IMD (Pestanal, Sigma-Aldrich, USA) solution in distilled water. The stock solution was placed in a glass bottle with an aluminium foil cover to protect from light exposure and was stored in a refrigerator (4°C) until used. Sucrose syrup solutions containing 0, 1.0 and 10.0 µg l −1 of IMD were freshly prepared and provisioned by feeders to the experimental colonies every third day throughout the experiment, which lasted a total of 10 consecutive weeks. This duration of exposure mimics field-realistic, long-term exposure to sublethal doses of IMD-treated crops that bumblebee may experience across the foraging season [68,69].
Each focal colony was weighed (to estimate colony growth) every week using a scale (Sartorius Corporation, USA; precision 0.01 g). We used a nest cage, identical to the ones used to house the colonies, installed with an empty plastic feeder (50 ml Falcon tube) and a 50 mm diameter Petri dish to tare the weight of the tested colonies before each measurement. We visually inspected the feeders every third day in order to assess sugar syrup consumption. We used the scale lines (2.5 ml resolution) displayed on their sidewalls for estimating the amount of liquid in each feeder. If the liquid level inside the feeder was approximately in the middle between measurement side-wall lines, the content was estimated as equivalent to the measurement line just below the liquid level plus 1.25 ml (e.g. liquid level in between 40-and 42.5-ml lines-we considered the final content as 41.25 ml). Pollen paste consumption was calculated by subtracting the weight of the Petri dish + pollen pellet at introduction and when removed from the colony 72 h later. Scattered pellets were collected and placed back on the Petri dish feeder before being weighed. We collected all newly emerging workers, gynes (young queens) and males, in each experimental colony on a near-daily basis (6-7 days a week), individually paint-marked them and estimated their body size, then returned them to their natal colonies. As an index for body size, we used the distance between the two tegulae (wing nod bases; termed below as 'IT span') that is highly correlated with other linear body-size measures such as marginal wing cell length and head width [64][65][66][70][71][72]. We measured body sizes over the course of 10 consecutive weeks, starting one week after the introduction of the bees to the new cages. We recorded the number of newly emerged workers on each day and used it as an index for colony population growth.

(d) Behavioural observations
We recorded the behaviour of queens and workers of the Control and IMD10 treatment colonies under dim red light, which bees do not see well [73,74], with minimal disturbance. A single observer recorded the frequency and duration of brood-care activities that included larval feeding, brood incubation, wax pot manipulation and scraping wax from pupal clumps [52,53,57,75]. These tasks were summed together to estimate the total percent of time a focal bee was engaged in brood care. Behaviors such as drinking sugar syrup, eating pollen, strolling in the nest, selfgrooming, fanning and inactivity were recorded as non-brood care-related activities [75][76][77]. The complete list of recorded behaviors and their description is summarized in the electronic supplementary information. We used a focal observation approach in which each observation session included recording queen behaviour for five consecutive minutes, followed by observations of two (individually tagged) workers for 5 min each, sequentially. Focal workers were randomly selected from the bees emerging in the first batch of workers. Observations were performed 3 days a week, over four consecutive weeks, for a total of 60 min per queen and 60 min per worker. All tested colonies were equally observed during morning (between 07:00 and 09:00) or late afternoon (between 16:00 and 18:00) sessions.

(e) Statistical analyses
Statistical analyses were performed using either R (v. 4.2.1; R Development Core Team 2022) or SPSS (IBM Corp., 2016, v. 24.0, Armonk, NY, USA), with p < 0.05 considered statistically significant. The effects of the tested IMD dietary concentrations on colony weight was analysed using the Generalized Estimating Equations test (G.E.E.) with gamma distribution (using SPSS). A first-order autoregressive correlation structure was also considered to account for a serial correlation across the repeated measures. For predictors within GEEs with statistically significant effect, we used pairwise post hoc Bonferroni tests. The influence of treatment on the total number of workers, gynes and males per colony was compared using the non-parametric Kruskal-Wallis test. We used generalized linear mixed models (hereafter 'GLMMs') to test the effects of IMD dietary concentrations on colony sugar syrup and pollen consumption, the body size of emerging bees, and queen and worker behaviour (using the lmer function from the package 'lme4' v. 1.1-30). First, we fitted four GLMMs with a Gaussian error structure and logit link function using syrup consumption (mL) and pollen consumption (g), and worker and gyne body size as response variables. For the models, we used IMD dietary concentration, colony age and their interaction as predictors, and we selected colony as random effect in order to control for the effect of repeated measures per colony. For the GLMM examining difference in gyne body size, we did not include colony age as a predictor variable because gynes emerge only toward the end of colony cycle. To test for the influence of IMD on queen and worker behaviour, we fitted four GLMMs analyses using total time inactive for queens, brood care events of queens, and brood care events of worker bees as response variables. We followed the same structure as above (i.e. same predictors, interactions and random effects) for consistency.
We controlled for type I errors by including all predictors and their interactions as random slopes. We used the 'car' package (v. 3.1-0) to assess multicollinearity in our multiple regression models assuming a threshold of 3 for variance inflation factors. We also visually verified that the residuals of our models were normally and homogeneously distributed by plotting them against fitted values as normal quantile and histogram plots. No model assumptions were violated. We compared full and null models including only random effects and offsets using likelihood ratio tests (anova function set to 'Chisq'). If the full model differed from the null model, we next tested which predictors account for the difference. To meet this goal, we used likelihood-ratio tests comparing the full model to reduced models in which one of the predictors was removed from the full model using drop1 function. For predictors with a statistically significant effect we performed a post hoc analyses using Tukey's HSD posthoc tests from the package 'emmeans' (version 1.8.1-1). Plots show either mean ± standard deviation (s.d.), mean ± standard error (S.E) or boxplots.

(d) Brood-care activities
Queens in the IMD10 treated colonies were recorded motionless more often (likelihood-ratio test: 2 Figure 1. Influence of chronic IMD treatment on the total number of adult bees emerging in a colony. Boxplots with different letters indicating a statistically significant difference in a Kruskal-Wallis ( p < 0.05) and a Dunn post hoc test ( p < 0.05), which were performed separately for each caste.

Discussion
Here, we reveal that exposure to sublethal levels of a neonicotinoid insecticide changes brood care behaviour of bumblebee queens, and the size distribution of offspring in young nests. Specifically (and counterintuitively), pesticide exposure led to the production of larger workers, but not larger gynes (which actually showed a trend towards smaller size in IMD-treated colonies). Our observations of worker and queen behaviour may help explain the production of larger-bodied workers in pesticide-treated nests. Queens in our insecticide-treated colonies exhibited decreased activity and reduced brood tending. Bumblebee queens have been shown to rear smaller-bodied offspring, relative to offspring reared by workers (e.g. B. terrestris [53]; B. impatiens [62]). Thus, a shift toward more worker brood care (relative to queen brood care) in the IMD-treated nests might have caused a shift toward the production of workers with a larger body size, which is determined during the larval stage in bumblebees [53,58,62]. Overall, these findings show complex influences of field-realistic, sublethal doses of a commonly used insecticide, operating specifically on the social biology of an ecologically and economically important pollinator species.
Given that the IMD-treated colonies consumed less food and produced fewer workers (which can bring food to larvae), it was unexpected to find that the workers emerging in these colonies were larger than in our control colonies. The body size of adult bumblebees is strongly influenced by the amount of food provisioned at the larval stage, and the overall quality of their care by tending adults [42,62,[78][79][80]. Although we did not assess queen fecundity directly, negative effects of the pesticide on queen fertility (reviewed by Camp & Lehmann [81]; see also [39,40,47,82]) may have led to a reduced number of offspring in the colonies. Brood care and other relevant factors, such as colony temperature and maintenance, are influenced by many factors including interactions between the queen, workers, and the developing brood [41,58,83]. Insecticides that modify adult bee behaviour or social interactions (such as imidacloprid [39][40][41][42]) may therefore lead directly or indirectly to changes in aspects of brood care that ultimately influence offspring size. For example, both IMD and the widely used herbicide Glyphosate impair bumblebee ability to thermoregulate their colonies, which can have a significant effect on the body size of emerging bees [41,83].
Queens of IMD-fed colonies were more commonly observed motionless (an effect also observed in B. impatiens [39]) and were specifically observed tending brood less often (figure 3b). Worker brood-tending activity decreased over time, with a sharper decrease in control colonies and lower levels during the first few weeks. This decrease over time may be explained by the increase in worker number, allowing the two focal workers to provide less care. In B. terrestris, larvae that are tended by queens develop faster, are commonly smaller, and are unlikely to develop into gynes [53,84]. These differences between the development of larvae tended by the queen or the workers support a self-organized model explaining the typical gradual increase in worker size along with colony growth and the switch from worker to gyne production toward the end of colony life [53,58]. The increase in the number of worker bees further allows the queen to reduce brood tending and increase egglaying activity, which may further increase colony population size, and dilute her influence on larval development [51,52]. Given that the queens of IMD-treated colonies provide less care for the larvae, which we suggest could escape at least part of her effects, ultimately developing into larger adults in the ad libitum fed laboratory colonies used for this study.
Our finding of larger-bodied workers in IMD-treated colonies differs from a field study in which B. terrestris colonies that were placed in clothianidin-treated oilseed rape fields had smaller workers and males [80]. Although we cannot exclude the possibility that these differences relate to the use of two different neonics, we believe that a likely explanation relates to differences between laboratory and field experiments. It is also worth noting that bumblebees in our study were only exposed to imidacloprid through sugar syrup, whereas in the field they may in addition be exposed to IMD in the pollen. Neonics influence various neurological functions such as orientation, learning and memory and circadian rhythms that influence foraging performance [85,86]. Thus, field colonies exposed to neonics may overall have a lower pollen and nectar supply, higher mortality leading to under-nourishment, and various compensation mechanisms that may increase metabolic costs and affect colony thermoregulation and overall homeostasis. There are also reports suggesting a general trend toward colonies producing smaller individuals in habitats subjected to anthropogenic influences [87,88]. Thus, although the effects on the queen may be similar in the field to what we found in our laboratory study, the undernourished field colonies cannot provision sufficient food to the larvae they rear, which therefore develop into smaller individuals than in neonic-free colonies. On the other hand, laboratory colonies invest significantly less energy in food gathering and processing (i.e. all workers are involved in in-nest activities), have an ad libitum food supply, and live in a constantly regulated environment, requiring them to devote less energy and time for regulating temperature and other aspects of the colony microenvironment (for example, see [41,83]). Additional studies in the laboratory and field are important for allowing us to uncouple foraging or colony homeostasis-related effects from influences on behaviour, physiology and social interactions inside the nest. Our findings corroborate the multifaceted effects of neonicotinoids on pollinator health and function and add to the small but growing cannon of studies showing that in social insects, these insecticides can have more nuanced, disruptive effects on intricate social processes. These include impacts on social interactions between adults and developing brood [41,83] that are essential for colony growth and fitness. We suggest that our observed effects on offspring body size are at least partially explained by IMD effects on queen behaviour specifically, which reduces her capacity to contact young larvae and manipulate their developmental programme [53,62]. The seemingly opposite effects of IMD on gyne body sizes may be related to the fact that gynes are produced late in the season when the colonies are larger and queens have ceased providing brood care, and thus the queen has little effect, if any at all, on larval development. The effects we observed should be further tested, given that our study is based on a limited number of colonies per group, and the results might be different under alternative food resource conditions, which can reveal novel pesticide effects [62,83]. Insecticide effects on larval development and ultimate body size may be specifically significant for bumblebees because body size is intricately connected with both caste determination and the division of labour among workers in this bee lineage. Thus, factors that influence female body size may hamper task performance, task allocation and overall colony performance [57,58,62,76,89,90]. Moreover, there is evidence that the body size of both gynes and males may affect their mating and reproductive success [72,[91][92][93]. Thus, insecticides with sublethal effects on the regulation of brood development may have an unexpectedly significant influence on colony fitness.
Data accessibility. All our data is provided in the paper or as electronic supplementary material [94].


d-Cysteine supplementation partially protects against ferroptosis induced by xCT dysfunction via increasing the availability of glutathione

Glutathione (GSH) is synthesized from three amino acids and the overall process is highly dependent on the availability of l-cysteine (l-Cys). GSH serves as an essential cofactor for glutathione peroxidase 4 (Gpx4), which reduces phospholipid hydroperoxides. The inactivation of Gpx4 or an insufficient supply of l-Cys results in the accumulation of lipid hydroperoxides, eventually leading to iron-dependent cell death, ferroptosis. In this study, we investigated the anti-ferroptotic properties of d-cysteine (d-Cys) under conditions of dysfunction in cystine transporter, xCT. l-Cys supplementation completely rescued ferroptosis that had been induced by the erastin-mediated inhibition of xCT in Hepa 1-6 cells. Upon d-Cys supplementation, the erastin-treated cells remained completely viable for periods of up to 24 h but eventually died after 48 h. d-Cys supplementation suppressed the production of lipid peroxides, thereby ferroptosis. The addition of d-Cys sustained intracellular Cys and GSH levels to a certain extent. When Hepa 1-6 cells were treated with a combination of buthionine sulfoximine and erastin, the anti-ferroptotic effect of d-Cys was diminished. These collective results indicate that, although d-Cys is not the direct source of GSH, d-Cys supplementation protects cells from ferroptosis in a manner that is dependent on GSH synthesis via stimulating the uptake of l-Cys.

F erroptosis is a recently characterized form of cell death that is dependent on intracellular ferrous iron. (1,2) The hydroxyl radicals produced in the Fenton reaction, which involve iron, are highly reactive and can cause an increase in the generation of lipid peroxides in membrane phospholipids that, in turn, cause ferroptosis by disturbing the integrity of the plasma membrane. The enzyme glutathione peroxidase 4 (Gpx4) uses glutathione (GSH), a major cellular antioxidant, to scavenge such lipid peroxides, and appears to play a pivotal role in protection against ferroptosis. (3) It has been reported that ferroptosis is associated with various pathological conditions, including cancer, neurodegenerative diseases, ischemic diseases, and inflammatory diseases. Because of this, the regulation of ferroptosis is expected to lead to novel therapeutic and preventive methods for the treatment of these conditions. (4) Cysteine (Cys) is an amino acid that contains a sulfhydryl group ( −SH), and its availability restricts the synthesis of GSH. Accordingly, Cys uptake is specifically required by many cells to prevent the onset of ferroptosis. (1,2) There is some evidence to show that Cys is directly taken up by cells from the environment, possibly via SLC1A4/5 (ASCT1/2) amino acid transporters. (5) However, in the oxidizing environment outside the cell, it is thought that free Cys is largely present in the form of cystine, a dimeric form of Cys linked by disulfide bond. (6) Cystine can be imported into cells via the amino acid transporter system x c − , a heterodimer of solute carrier family 7 member 11 (SLC7A11, xCT) and solute carrier family 3 member 2 (SLC3A2, 4F2hc), (6) on the cell membrane. When the x c − system is inhibited by erastin, or when extracellular Cys/cystine is degraded using an engineered enzyme, cyst(e)inase, ferroptosis is induced in diverse cancer cell lines and model animals. (7)(8)(9) In a previous study, we reported that the inhibition of GSH synthesis alone by buthionine sulfoximine (BSO) is not sufficient to induce ferroptosis in some types of cells, (10) suggesting that free l-Cys functions independently in protecting against ferroptosis. It has been reported that d-Cys is not a biosynthetic precursor of GSH but it has also been shown that hydrogen sulfide (H 2 S) and its storage form (bound sulfur) are synthesized from d-Cys as well as from l-Cys in vivo. (11)(12)(13) Both d-Cys and l-Cys contain free −SH groups, but it has not been established that d-Cys functions in antioxidant defense against ferroptosis. In the current study, the anti-ferroptotic properties of d-Cys were examined under conditions of xCT dysfunction. The findings show that d-Cys supplementation partially suppresses ferroptosis by promoting the cellular uptake of l-Cys and the subsequent synthesis of GSH.
Evaluation of the cytotoxicity of cells. Cells were seeded at a density of 1 × 10 5 cells/ml of medium in 35-mm tissue culture dishes (2 ml/dish) and incubated overnight. Cytotoxicity was determined by means of a lactate dehydrogenase (LDH) assay, as described previously. (15) The reaction mixture contained 20 μl of culture medium, 0.3 mM NADH, 1 mM sodium pyruvate, and 200 mM sodium phosphate buffer, at pH 7.4 in a total volume of 100 μl. Initial activities were calculated from the rate of disappearance of NADH during the starting linear phase of the reaction by monitoring the absorbance of the medium at 340 nm.
Evaluation of the viability of cells. Cells were seeded at an initial density of (1.0 × 10 5 /ml). Cell viability was determined using a CellTiter-Blue ® Cell Viability Assay (Promega, Madison, WI) according to the manufacturer's instructions. Fluorescence intensity was measured using a microplate reader Varioskan Flash (Thermo Fisher Scientific) with an excitation wavelength  of 560 nm and an emission wavelength of 590 nm. Fluorescence values were then normalized with respect to cells and are considered to be 100%.

Measurements of cystine, GSH, and Cys levels by liquid
chromatography-mass spectrometry analyses. Sample preparation and liquid chromatography (LC)-mass spectrometry (MS) were performed by previously reported methods. (16) For measurement of extracellular cystine levels, equal volumes of conditioned medium and 50 mM ammonium bicarbonate containing 20 mM N-ethylmaleimide (NEM), pH 8.0, was mixed, boiled for 3 min and then incubated at room temperature for 10 min. The reactants were mixed with an equal volume of methanol and an equal volume of methanol containing a 10 μM internal standard. After incubation on ice for 2 h, the mixture was centrifuged at 15,000 rpm for 15 min at 4°C and the resulting supernatant was used for LC-MS analysis. For the measurement of intracellular GSH and Cys, cells were lysed by treatment with 50 mM ammonium bicarbonate containing 20 mM NEM, pH 8.0, boiled for 3 min and then incubated at room temperature for 10 min. The resultant lysate was mixed well with an equal volume of methanol and an equal volume of methanol containing a 10 μM internal standard. A two-fold volume of chloroform was added to the mixture with vigorous mixing. After centrifugation at 15,000 rpm for 15 min at 4°C, the upper phase was used for the LC-MS analysis. The samples were applied to a Sequant ZICpHILIC (5 μm, 2.1 × 150 mm; Merck, Darmstadt, Germany), using an Ultimate 3000 LC system (Dionex, Sunnyvale, CA), equilibrated with 10% mobile phase A (20 mM ammonium bicarbonate, pH 9.8) and 90% mobile phase B (100% acetonitrile), and eluted at a flow rate of 0.15 ml/min with a gradient of 90-42% mobile phase B in 20 min and then maintained at 42% B in 10 min. The LC system was composed by a WPS-3000 TRS autosampler, a TCC-3000 RS column oven, and an HPG-3400RS quaternary pump (Dionex, Sunnyvale, CA) and connected to a quadrupole Orbitrap (Q-Exactive, Thermo Scientific) mass spectrometer equipped with a heated electrospray ionization source. Standard curves for cystine, GSH-NEM, and Cys-NEM were generated in the ranges of 0.1-3,000 μM, 0.01-300 μM, and 0.01-300 μM, respectively. The resulting curves showed linearity in these ranges for the authentic compound level. System control, data acquisition, and quantitative analysis were performed with the Xcalibur 2.2 software (Thermo Fisher Scientific). Flow cytometry. For lipid peroxidation assays, cells were incubated with 10 μM C11-BODIPY 581/591 (Thermo Fisher Scientific) for 30 min following the manufacturer's instructions and then washed with PBS. After trypsinization, the cells were collected and subjected to flow cytometry (FACSCanto TM II, BD Biosciences, Tokyo, Japan).
Statistical analysis. Statistical analyses were performed using the GraphPad Prism ver. 6.0 for Mac (GraphPad Software, San Diego, CA). A p value of less than 0.05 was considered to be significant.  medium. The erastin treatment resulted in a decreased cell viability in Hepa 1-6 cells (Fig. 1A). When l-Cys was added to the medium, the cell viability was improved and this improvement was concentration-dependent. Thus, an insufficient supply of Cys to the cells was found to be responsible for the erastininduced cell death. As we expected, d-Cys also improved cell viability to a certain extent, although this was less effective than l-Cys.

Results
We next measured LDH activity, a hallmark of necrotic cell death, in the culture supernatant to evaluate the properties of this cell death (Fig. 1B). While the treatment of the cells with erastin elevated the LDH activity in the medium, erastin-induced LDH release was completely suppressed by l-Cys supplementation during the 48 h culture period. When d-Cys was added, the Hepa 1-6 cells remained completely alive for periods of up to 24 h, which was also confirmed by phase-contrast microscopy ( Fig. 1C), but the cells eventually died 48 h later. Thus, d-Cys was protective for limited periods under conditions of xCT inhibition.
We next evaluated the levels of lipid peroxidation products, which are considered to be a characteristic of ferroptosis among the various types of cell death, using C11-BODIPY 581/591 , a sensitive fluorescent probe for the detection of lipid peroxidation products. At the 24-h time point, the fluorescent intensity was elevated in the cells that had been treated with erastin, indicating the augmentation of lipid peroxidation in the cells. Consistent with the results of the cell death assay, the increased fluorescent intensity was also completely suppressed by the addition of d-Cys as well as l-Cys at this time point (Fig. 1D).
We also examined the effect of d-Cys against ferroptosis induced by cystine-free medium. As a result, LDH activity in the medium increased when Hepa 1-6 cells were cultured in cystine-free medium, whereas the addition of l-Cys completely suppressed ferroptosis during the 48 h culture period ( Fig. 2A and B). On the other hand, the addition of d-Cys was able to somewhat inhibit the cell death of Hepa 1-6 cells up to 24 h, but the cells eventually died 48 h later. Furthermore, the increase in lipid peroxidation caused by cystine deprivation was completely inhibited by l-Cys supplementation, but the protective effect of d-Cys was less pronounced (Fig. 2C). These results indicate that the protection by d-Cys against ferroptosis was less effective in a Cys/cystine-deficient environment compared to that under xCT inhibition by erastin.
To gain insights into the molecular mechanism responsible for this, we then measured the intracellular levels of total Cys, which were the sum of the levels of l-Cys and d-Cys, and GSH (Fig. 3), which were produced from l-Cys only, (11,12) by means of LC-MS. Their concentrations were markedly decreased after 12 h of the erastin treatment while l-Cys supplementation drastically restored them (Fig. 3A and B). Under xCT inhibition by erastin, the addition of d-Cys improved intracellular levels of Cys to a certain extent and also intracellular GSH levels were maintained.
The addition of l-Cys and d-Cys to the cystine-deprived medium both increased the amount of intracellular Cys (Fig. 3C) but the amount of intracellular GSH increased only when l-Cys was added (Fig. 3D). The levels of extracellular cystine, the sum of the l-Cys dimer, the d-Cys dimer, and chimera of l-Cys and d-Cys, were comparable between the l-Cysand d-Cyssupplemented medium (Fig. 3E and F). Collectively, these results indicate that intracellular levels of Cys and GSH were maintained until a certain time even by d-Cys supplementation, leading to protection against ferroptosis.

D-Cys protects xCT-KO MEFs from death induced by the
withdrawal of β-ME from the culture medium. To further confirm the anti-ferroptotic action of d-Cys, we used embryonic fibroblasts established from xCT-knockout mice (xCT-KO MEFs). Since xCT-KO MEFs do not produce xCT, the cells die when cultured in standard culture medium without supplementation with β-ME, which can rescue the death of xCT-KO MEFs. (14) After removing β-ME, cell death occurred, as evidenced by an increase in LDH levels in the culture medium (Fig. 4A). When l-Cys was added instead of β-ME, cell death was completely suppressed during the 48 h treatment, suggesting that the l-Cys is taken up via corresponding amino acid transporters other than xCT. On the other hand, d-Cys suppressed cell death until 24 h, but the protective effect was lost after 48 h, indicating that d-Cys rescued xCT-KO MEFs from ferroptosis but only at an earlier period of incubation. We next determined the intracellular levels of Cys and GSH in xCT-MEFs and observed significant decreases in their levels under β-ME-free conditions ( Fig. 4B and C). Supplementation with l-Cys or d-Cys significantly restored both Cys and GSH levels to similar extents between the two groups of cells.

D-Cys supplementation protects cells from ferroptosis in
a GSH synthesis-dependent manner. Finally, to examine the protective mechanism of d-Cys, we treated Hepa 1-6 cells with a combination of erastin and BSO, a compound that inhibits the action of γ-glutamylcysteineligase (GCL), the rate-limiting enzyme for GSH synthesis (Fig. 5). Consistent with our previous report, (10) the BSO treatment alone was not sufficient to result in lethality in Hepa 1-6 cells, as evidenced by an LDH assay. We found that the simultaneous treatment with BSO and erastin ( Fig. 5A and B) or the pretreatment with BSO followed by treatment with erastin (Fig. 5C)  these results suggest that d-Cys supplementation protects cells from ferroptosis in a GSH synthesis-dependent manner, even though d-Cys cannot be used for the GSH synthesis.

Discussion
In the current study, we found that ferroptosis that is induced by the inhibition of xCT by erastin was suppressed not only by the addition of l-Cys but also by the addition of d-Cys (Fig. 1). Although d-Cys is not a direct source of GSH synthesis, d-Cys supplementation resulted in both intracellular Cys and GSH levels being temporarily maintained under xCT dysfunction (Fig.  3). On the other hand, under extracellular cystine deprivation, d-Cys supplementation did not increase the intracellular GSH level, and the protection by d-Cys against ferroptosis was less effective (Fig. 2). These observations confirmed pivotal action of preserved GSH, which was only temporarily performed by d-Cys, for the cytoprotective action. We also found that d-Cys suppressed ferroptosis in xCT-KO MEFs after removing β-ME from the medium (Fig. 4), which eliminates the possible involvement of xCT in the d-Cys-mediated protection in erastin-induced ferroptosis. Finally, we found that GSH synthesis was essential in this d-Cys-mediated suppression of ferroptosis (Fig. 5). These collective data clearly indicate that the Cys-GSH axis is involved in the d-Cys-mediated protective action against ferroptosis that is transporter. (6) In fact, this disulfide exchange reaction is partly responsible for the protective role of β-ME in maintaining xCTdeficient cells. (14,17,18) β-ME reacts with l-cystine to produce a mixed disulfide between l-Cys and β-ME via thiol-disulfide interchange reactions, which results in one l-Cys being released. Both the released l-Cys and a mixed disulfide linking l-Cys and β-ME can be transported into the cell via membrane transporters for neutral amino acids. The mixed disulfide is then reduced back to l-Cys and β-ME after entering the cells. β-ME is exported out of cells and reacts with l-cystine in the extracellular milieu again. This β-ME-mediated cyclic action allows cells to maintain sufficient levels of l-Cys and consequent GSH levels under culture conditions even in the case of xCT-deficient cells. (18) However, because the measurement method used in our study does not distinguish between Cys isomers, it is not possible to conclude that the chimeric cystine consisting of Cys isomers is transported into the cells. In the case where the chimeric cystine is not incorporated into cells, the d-Cys-mediated l-Cys supply to the cells would be less efficient compared to the β-MEmediated one. Our results show that rescuing the action of d-Cys was effective but only within 24 h, and suggest that Hepa 1-6 cells were unable to incorporate the chimeric cystine. On the other hand, we cannot eliminate other possibilities that involve the function of sulfur derived from d-Cys because the inhibition of GSH synthesis alone does not cause ferroptosis in some cells, including the Hepa 1-6 cells used in this study. (10) For example, both l-Cys and d-Cys can serve as a precursor for the biosynthesis of hydrogen sulfide (H 2 S), (13) which has ability to reduce the disulfide bonds of l-cystine, resulting in the formation of two free l-Cys molecules. (19) d-Cys is first converted to 3mercaptopyurvate by d-amino acid oxidase and then to H 2 S by the action of 3-mercaptopyrvate sulfurtransferase in some organs. Although H 2 S was not measured in our study, we consider that this mechanism is unlikely here because the enzymes responsible for the conversion of d-Cys to H 2 S are localized in only limited tissues. l-Cys is a precursor for synthesizing not only proteins, GSH, and H 2 S but also some primary compounds, as for example, sulfur-iron complex, (20) coenzyme A (CoA), (8,21,22) and Cys persulfides. (23) Whether d-Cys is utilized in their syntheses will require further investigation and there remain unanticipated mechanisms for the suppression of ferroptosis by d-Cys. In this study, d-Cys was found to partially protect cells against ferroptosis that is induced by xCT inhibition or the ablation of the xCT gene. The protective mechanism appears to be attributable to the l-Cys that is released through the replacement of one l-Cys in l-cystine with d-Cys by a disulfide exchange reaction. In fact, in the absence of extracellular l-cystine, d-Cys supplementation did not increase intracellular GSH levels, and the anti-ferroptotic action was limited. The preserved l-Cys is then incorporated into cells via neutral amino acid transporters and is used to synthesize GSH, leading to protection against ferroptosis. Regarding the mechanism responsible for this process, the chimeric cystine composed of Cys isomers will need to be identified.


Traditional Uses, Phytochemistry, and Bioactivities of Mesosphaerum suaveolens (L.) Kuntze

Mesosphaerum suaveolens (L.) Kuntze is a species widely used traditionally in the treatment of ailments, such as stomach pain, hemorrhoids, cough, verminosis, ulcer, liver disease, fever, influenza, nasal congestion, and inflammation. This review aims to provide a survey of available information on seven international electronic databases (Google Scholar, Medline, ResearchGate, Web of Science, Scopus, Science Direct, and PubMed) about botanical aspects, traditional uses, phytochemistry, and biological activities of M. suaveolens. Mesosphaerum suaveolens is a tropical America native species, but it can be found in several parts of the world as a ruderal plant. The species is the most studied species of the genus Lamiaceae due its phytochemical aspect, especially regarding the chemical composition of its essential oil. Besides the essential oils, M. suaveolens is a source of numerous secondary compounds such as triterpenes, diterpenes, and phenolic compounds, which are related to its biological activities, such as allelopathic, antibacterial, antifungal, insecticidal, and larvicidal activities as described in the literature.

Introduction
Plant species, with medicinal properties that have always been part of human life, are being used both for the treatment of diseases as for food. For the treatment of diseases, they are accessible and culturally accepted, so their use is popular since ancient civilizations [1,2]. e Lamiaceae family is one of the most diverse and widespread in terms of the ethnomedicinal value and variety of plants with biological and medical applications [3][4][5][6][7][8]. Regarding the genus Mesosphaerum, previous studies demonstrate the ethnomedicinal and pharmacological importance of some species that belong to it, such as Mesosphaerum sidifolium (L'Hérit.) Harley & J.F.B. Pastore, used to treat stomach disorders and headaches, as well as being used as an expectorant, carminative, and tonic. is species possesses in vivo antitumor activity against Ehrlich ascites carcinoma cells causing growth inhibition by inducing cell cycle arrest, besides not showing cytotoxicity [9]. Another species with several bioactivities is Mesosphaerum verticillatum (syn. Hyptis verticillata Jacq.) with anti-inflammatory, antimicrobial, and anticancer potentials, among other reports. Ethnomedicinal uses of this plant include cough, colds, asthma, fever, tonsillitis, uterine fibroids, bronchitis, and gastrointestinal problems [5].
In Northeast Brazil, the use of plant species as therapeutic resources is widespread, and one species present in this region is Mesosphaerum suaveolens (L.) Kuntze, known as "bamburral," "erva-canudo," or "alfazema-brava" [10]. Its leaves are mainly used to treat respiratory diseases (asthma, bronchitis, colds, and flu) and diseases related to the gastrointestinal tract [1]. Such medicinal uses are related to the chemical heterogeneity arising from the secondary metabolism of the species, a recurrent characteristic in species of the Lamiaceae family [6].
Several works in the literature indicate that M. suaveolens presents a high biotechnological potential, mainly regarding its essential oil [10]. In addition, a large number of studies have emphasized the biological activities of the essential oil and extracts of this species against pathogenic microorganisms to humans [6]. Taking into account that M. suaveolens is a medicinal species widely studied by the scientific community, the main objective of this work was to make a general review of the botanical aspects, traditional uses, phytochemistry, toxicity and biological, and pharmacological activities.

Materials and Methods
Methodologically, it was used the keywords "Mesosphaerum suaveolens" and its synonym "Hyptis suaveolens" associated to the terms "biological activity," "bioactive," "ethnomedicinal use," "traditional use," "ethnobotany," "ethnopharmacology," "toxicity," "natural products," "phytochemistry," and "allelopathy" to collect information available on Google Scholar, Medline, ResearchGate, Web of Science, Scopus, ScienceDirect, and PubMed databases. e consideration insertion criteria of the articles were as follows: full article only, articles written in English and/or Portuguese languages, and all available and opened access articles, with no time limit determined.
It was obtained 190 articles dated between 1971 and 2021 which were grouped into some categories. (1) Botanical aspects, with information on description, classification, and geographical distribution; (2) phytochemistry; (3) ethnobotany; (4) biological activities; and (5) pharmacological activities. e trial process (collecting of the articles, reading of the abstracts, and checking the insertion criteria) took three months, and all the selected articles had been read completely and summarized in a table with the isolated chemical constituents and their respective biological activities.

Botanical Aspects: Description, Classification, and
Distribution. Mesosphaerum suaveolens (L.) Kuntze is an herbaceous plant belonging to the Lamiaceae family. e word "mesosphairon" comes from the Greek and Latin "mesosphaerum," meaning "a type of tuberose with mediumsized leaves," while its specific epithet suaveolens, means "with a sweet fragrance" due to the aroma of essential oils exhaled by the trichomes present on its leaves [11,12].
Morphologically, M. suaveolens is an erect herb or subshrub that measures up to 2 m in height. Its photosynthetic quadrangular stem is hairy with closely spaced branches and nodes. It has oval leaves, serrate or cordate margin, pilose limb, acute apex, and obtuse base with opposite crossed phyllotaxis. e petioles are short, canaliculate, as are its stems. Its inflorescences consist of up to 20 flowers located around the nodes and near the leaf axils. e flowers are pedunculate, with a persistent, tubular calyx, and 5 pointed sepals. e corolla is also tubular with five lilac petals, and the lobes are evident. Its fruits are dry, indehiscent, and uniseminated, originating from a bicarpellate gynoecium. Such fruits originate dimorphic seeds, two per fruit. Morphologically, such diaspores are elongated with dorsoventral flattening, longitudinal median ridge, starting near the hilum and extending to the apex of the seed with retusa boundary with black coloration (Figure 1) [16][17][18].
As for the geographical distribution, M. suaveolens is native to tropical America; however, as it is ruderal, it ended up invading natural ecosystems in tropical and subtropical regions of the globe, so that, due to this widespread occurrence, the species is considered a pantropical ruderal species [19][20][21][22]. In Brazil, M. suaveolens is present in almost the entire territory [23].

Phytochemistry.
Mesosphaerum suaveolens is an important source of essential oils, alkaloids, flavonoids, phenols, saponins, triterpenes, and sterols [13,24]. e essential oil of this species, obtained exclusively from its leaves, has already been chemically characterized in several studies. However, since this species exhibits a high level of genetic polymorphism and allows adaptation to changes in environmental characteristics, high variability in the composition and content of the major constituents (>20%) has been found [25]. In M. suaveolens extracts, terpenoids had a great predominance (mono, di, tri, and sesquiterpenes) ( Table 1)  (Table 1). Among the diterpenes, suaveolic acid stood out with recognized antimicrobial and allelopathic action [26]. Furthermore, phenolic acids, phenylpropanoids, flavonoids [10,27], and fatty acids [28,29] were also identified in different parts of M. suaveolens (Table 1).
In the Asian continent, more specifically in India, the leaves, stems, inflorescence, and roots are used to treat urinary calculi [72], stomach pain [73], healing, itching [74], boil, eczema, diabetes [75], pneumonia [76], and fever [77]. Besides that, the seeds of M. suaveolens are used to treat gynecological disorders such as menorrhagia, leucorrhea, and rheumatism [78,79]. e fresh poultice of the leaves is applied to snake bites, wounds, and mycoses [80], while the paste of the fresh leaves is also indicated for skin diseases [81].
In South Asia, in Bangladesh, traditional communities use the seeds in juice preparations to treat constipation and weakness [82,83]; in addition, the seeds are consumed along with roots of Bombax ceiba to treat gonorrhea [84,85], and the paste of the leaves is used to treat skin infections [86]. In Togo, the leaves of the species are spent in decoction form for the treatment of gynecological disorders [87], while in ailand, the decoction of the roots is indicated in cases of food poisoning [88]. On the African continent, more specifically in Benin and Nigeria, the whole plant of   Evidence-Based Complementary and Alternative Medicine 5  Evidence-Based Complementary and Alternative Medicine 7  Evidence-Based Complementary and Alternative Medicine 9 M. suaveolens is used for the treatment of candidiasis and as a blood tonic [89,90]. From a veterinary point of view, M. suaveolens has also been used for the treatment of diseases in animals. Such use is reported in India for the treatment of inflammation in cattle, with the juice of the leaves being applied to the animal's eyes [91]. In Brazil, the species is employed against diarrhea [92]. In the African continent, more specifically in Kenya, the aerial parts of the plant are utilized as a repellent for the mosquito Anopheles gambiae Giles, 1926 (Diptera: Culicidae) [93,94].

Biological Activities
3.4.1. Allelopathic Activity. According to Sharma et al. [95], after the establishment of M. suaveolens in an area, it becomes evident that the species imposes a profound impact on the local vegetation, as the number of species, richness, diversity, and uniformity is severely reduced. Although M. suaveolens is native to the Brazilian territory, it is distributed in different ecosystems, such as Caatinga, a seasonally dry tropical forest [20].
Mesosphaerum suaveolens produces numerous seeds of rapid germination and subsequent growth and thus manages to occupy and dominate environments because of its allelopathic action [96]. Islam et al. [26], for example, isolated suaveolic acid from M. suaveolens and demonstrated in bioassays that this diterpene exhibits allelopathic action, interfering with the growth of the caulicle and radicle of . eir extracts present allelopathic action against Echinochloa crusgalli (L.) P. Beauv. [97], Sorghum vulgare Pers., Raphanus sativus L., and Lactuca sativa L. [98].
Allelochemicals present in the species have been reported to act by causing oxidative stress, reduction in chlorophyll content, and inducing the formation of chromosomal aberrations [99,100]. Such damage may occur in response to the synergistic action of the constituents.
In addition to heterotoxicity, M. suaveolens has been found to exhibit autotoxicity; however, its constituents affect other species more than itself [101]. us, the low amounts of allelochemicals released by M. suaveolens affect the ecological succession of other species, but do not affect the species itself as much.
Despite reports of the allelopathic action of M. suaveolens, it is worth noting that most of these studies were conducted under laboratory conditions and with extracts of the plant, so these actions do not match the allelopathic actions found in the environment.
us, it is necessary to conduct studies that simulate as much as possible the natural conditions, to affirm whether one species can affect another. Only Kapoor [99] evaluated the allelopathic action of M. suaveolens in conditions similar to those found in the environment, demonstrating in fact that the species has allelopathic action on Parthenium hysterophorus L. (Asteraceae).
M. suaveolens has allelochemicals from its secondary metabolism, which compromise the structure and plant diversity [102].

Antimicrobial Activity.
Teas of M. suaveolens are used to treat diseases related to the gastrointestinal and respiratory tracts [1], so numerous researchers have hypothesized that the species exhibits biological activity against strains of pathogenic microorganisms.
Cyrille et al. [103] evaluated the antibacterial action of the hydroethanolic extract (70%) of the leaves and found that the species presented low antibacterial activity since an MIC of 3.12 mg/ml was observed against Staphylococcus aureus (Rosenbach, 1884) (Staphylococaceae) ATCC 25923 and Pseudomonas aeruginosa (Schroeter, 1872) (Pseudomonadaceae) ATCC 27853 strains. It is worth noting that MIC values obtained above 1 mg/ml (1000 μg/ml) do not reflect clinically notable activity (Van Vuuren, 2008) ( Table 2). e works evaluating the antimicrobial action of the species highlight the use of the volatile terpenes (essential oils) of the leaves (  Evidence-Based Complementary and Alternative Medicine  Evidence-Based Complementary and Alternative Medicine 13  [126] 14 Evidence-Based Complementary and Alternative Medicine   Table 2).

Insecticidal Activity.
Popularly, in Kenya, the aerial parts of M. suaveolens are burned to repel mosquitoes of the species Anopheles gambiae [84,94]. Subsequently, other researchers have highlighted that the oil has biological action against various insects, such as A. gambiae itself (Table 3).

Repellent Activity.
Besides causing mortality in insects, the products of M. suaveolens, especially the essential oil of the leaves, can repel insects and arachnids of public health and economic interests ( Table 4). As for Diptera, the essential oil by fumigation and the fresh leaves by contact were able to repel mosquitoes of the species Anopheles gambiae, malaria vector, with the former showing an effective rate of 98% repellency at a low concentration (6%) [152], while the leaves when rubbed on the body had a rate of 66.5% [153]. Besides this Diptera, Aedes aegypti vector of arboviruses, such as dengue, yellow fever, chikungunya, and zika, was also repelled when in contact with the ethyl acetate extract of the leaves.

Other Biological Activities (Antiarachnidic, Antiparasitic, and Molluscicides).
In addition to the aforementioned activities, the herbaceous species present bioactive compounds against other biological organisms. Among these were highlighted parasitic organisms of humans, as reported by Shittu et al. [180], in which evaluating the trypanocidal action (Trypanosoma brucei brucei) in vivo of gold nanoparticles from M. suaveolens demonstrated that the species can cause a complete clearance of the parasite after seven days of infection. As well as in insecticidal action against malaria vectors (Anopheles spp.), M. suaveolens exhibits antiplasmodial activity (Plasmodium falciparum 3D7) [45,46,181]. Noronha et al. [181] evaluated the antiparasitic Leaf essential oil Acute toxicity Aedes aegypti (CL 50 139.07 μg/ml) [174] [179] Evidence-Based Complementary and Alternative Medicine action of the methanolic extract of M. suaveolens leaves and observed that the natural product has an IC 50 of 3.906 μg/ml against chloroquine-sensitive Plasmodium falciparum strains. In the study by Ziegler et al. [45], the researchers isolated a diterpene (dehydroabietinol) from the leaves and observed that the compound was able to inhibit 50% of parasite growth at a low concentration of 7.3 μg/ml. Similarly, Chukwujekwu et al. [46] demonstrated that another diterpene (13α-epi-dioxiabiet-8(14)-en-18-ol) isolated from the same organ showed an IC 50 of 0.11 μg/ml, against P. falciparum D10, being of great clinical interest.
Research involving the extracts and essential oil of M. suaveolens focus on insecticidal activities, so only one work aimed to evaluate the bioactive potential against Arachnida species [182]. In this study, the authors demonstrated that the essential oil of the leaves at the concentration of 31.3 mg/ml can cause 50% mortality of Rhipicephalus (Boophilus) microplus for engorged females, while for juvenile forms of the tick, CL 50 is 51.6 mg/mL, demonstrating that females are more susceptible to the oil.
Salawu and Odaibo [182] evaluated the molluscicide action of the ethanolic extract of M. suaveolens against Bulinus globosus, found that the product presents both lethality in adult individuals (LC 50 77 μg/ml), and also presents an ovicidal potential (LC 50 614 μg/ml). us, it demonstrates that the species is a source of compounds of pharmacological interest for the development of natural antioxidant products.

Pharmacological
Agarwal [183] showed that the methanolic extract of M. suaveolens leaves has potent antioxidant activity evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method with IC 50 value � 40.91 μg/ml, and there is about 69% free radical inhibition capacity at the highest concentration (100 μg/ml). It was also found that the percentage of inhibition is concentration dependent, the higher the concentration the greater the inhibition of free radicals, and that the main phytochemicals involved in this activity may be phenols and flavonoids. e methanolic extract also demonstrated antioxidant activity with assays other than DPPH, such as ferric reducing antioxidant power (FRAP) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) [184].
Other studies have also evaluated the free radical scavenging potential by the DPPH method. Gavani and Paarakh [185] [186]. Such results are attributed to the phenolic compounds present in such extracts.
In the work of Priyadharshini and Sujatha [188], four types of leaf extracts were evaluated; in the DPPH assay, three of the extracts had significant results, the best of them being the ethyl acetate extract with a percentage of inhibition (IC 50 � 137 μg) very close to that of the standard used, ascorbic acid (IC 50 � 127 μg). In this same study, the acetate extract also stood out with other tests; in the superoxide anion radical scavenging assay, the IC 50 value � 22.94 μg, and the standard had a value of 21.47 μg.
In addition to leaves, antioxidant potentials of other organs such as flowers were investigated in the study by Banerjee and De [189]. However, the results were not promising, as the extract of the reproductive parts exhibited an IC 50 of 1690.21 μg/ml against DPPH. Antioxidant studies involving M. suaveolens are not restricted to extracts only. Nantitanon et al. [126] evaluated the effect of the essential oil from the leaves; however, it showed low potential to reduce free radicals (IC 50 of 3.7200 mg/ml). Hsu et al. [190] demonstrated that the seeds also exhibit antioxidant action, as they showed moderate inhibitory activity against xanthine oxidase. In a study conducted by Lima et al. [191], it was observed that the essential oil from M. suaveolens leaves showed moderate Fe 2+ chelating activity at the concentration of 480 μg/ml.

Healing Potential.
e healing potential of M. suaveolens leaves was evaluated by three types of extracts (petroleum ether, ethanolic, and aqueous) in different wound models (excision, incision, and healing) using albino Wistar rats [192]. In this study, it was revealed that the extract using petroleum ether had the greatest significant effect on wound healing in murine wounds. Shirwaikar et al. [193] evaluated the ethanolic extract on these same three types of wounds, also obtaining significant results which were justified by the free radical scavenging action of this species.

Neuroprotective Activity.
Ghaffari et al. [184] determined the neuroprotective potential of the methanolic extract of the aerial parts of M. suaveolens on mouse N2A neuroblastoma cells, in which they observed that the natural product inhibits hydrogen peroxide (H 2 O 2 )-induced neuronal death. e authors justified this effect by the fact that the extract can regulate the activation of antioxidant and protective genes of the nerve cells.
ese results are promising, and the methanolic extract can be employed to treat stress-induced neurodegeneration. [194] evaluated the anti-inflammatory potential of ethanolic extract of the leaves against inflammation induced by carrageenan in albino rats; the extract showed significant results when compared to standard ibuprofen; this result is justified by the good antioxidant activity of this extract. In the study by Grassi et al. [42], two diterpenes (C 20 ) isolated from M. suaveolens, suaveolol and methyl suaveolate, showed anti-inflammatory potential when evaluated regarding the reduction of ear edema in rats. In such research, it was observed that the compounds reduced inflammation with ID 50 � 0.71 μmol/cm 2 (dose giving 50% edema inhibition) for suaveolol and ID 50 � 0.60 μmol/cm 2 ; however, despite the pharmacological effect, the results were only two to three times less active than the standard drug used in the study indomethacin (ID 50 � 0.26 μmol/cm 2 ).

Anti-Inflammatory Activity. Shenoy and Shirwaikar
3.5.5. Antiulcer Activity. Mesosphaerum suaveolens leaves are popularly used for the treatment of gastric ulcers; however, no active ingredient had been identified. e first study evaluating such an effect was by Vera-Arvaze et al. [43], in which such authors isolated the diterpene suaveolol from the leaves and evaluated it against an induced experimental model; the results presented indicated that this compound had a gastroprotective effect of more than 70%.
One year after the publication of the mentioned study, Jesus et al. [195] using the ethnopharmacological approach of M. suaveolens evaluated its antiulcer potential through the ethanolic extract and its fractions. e results for all products had high significance, p > 0.001, with the hexanic fraction of the extract being the most effective with 74% inhibition of induced gastric ulcer at a dose of 500 mg/kg. 3.5.6. Antidiarrheal Activity. Although M. suaveolens is popularly used to treat gastrointestinal disorders such as diarrhea, only the work of Shaikat et al. [196] evaluated its antidiarrheal potential. e researchers prepared ethanolic extracts of the leaves and based on the popular use, evaluated against an experimental model of diarrhea induced in mice; the results obtained show that this species presents compounds with the antidiarrheal effect (p > 0.001) that may be acting in an isolated or synergistic way.

Antihyperglycemic Activity.
e ethanolic extract of M. suaveolens leaves was evaluated in the in vivo experimental model of streptozotocin-induced diabetes. e extract at doses of 250 and 500 mg/kg bodyweight was administered orally over 21 days; at the end of the treatment, a decrease in the levels of triglycerides, total cholesterol, and low-density lipoprotein can be seen; these results indicate that this species has significant antidiabetic activity [197].
3.5.8. Hepatoprotective Effect. Ghaffari et al. [198] induced damage to livers of Wistar rats using carbon tetrachloride (CCl4) and subsequently administered doses of 50 and 100 ml/kg of methanolic extract of the aerial parts of M. suaveolens. e results were promising, demonstrating that the extract has a hepatoprotective effect, which can be explained by the antioxidant potential of the product, also demonstrated in the study.
3.5.9. Toxicity. A topical cream based on M. suaveolens essential oil was prepared based on its popular use in ailand, where they pointed out further investigations related to its toxicity in humans. is study was conducted on Wistar rats for 28 days under 3, 10, and 30% concentration of essential oil. e results showed us that concentrations of 3 and 10% did not cause a statistically significant dermal toxicity unlike the 30% concentration in which some female rats presented signs of erythema on its shaved dorsal skin between 11 and 14 days after the cream application [199].

Conclusions
It is evident that M. suaveolens is the most studied species of the genus, traditionally used in the new and old world to combat several diseases. Chemically, the species is much investigated, mainly about the composition of its essential oils that can vary according to the locality of occurrence. Mesosphaerum suaveolens also behaves as ruderal, and its success may be due to the release of allelochemicals. e species also presents a biotechnological potential corroborated by the remarkable activity against pathogenic microorganisms, insects, and other arthropods that transmit diseases. Finally, the pharmacological applications of the species are highlighted, especially the antioxidant action found in several organs of the species.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.